Methods for purifying bacteriophage and products of manufacture containing endotoxin-free bacteriophage preparations

Abstract

In alternative embodiments, provided are compositions, including products of manufacture and kits, and methods, for purifying bacteriophage. Provided herein is are practicable methods, or protocols, that are “Good Laboratory Manufacturing Practice” (GLMP), for phage isolation, selection, liter-scaled cultivation, and purification. In alternative embodiments, GLMP protocols as provided herein employ membrane filtration processes to yield at least about 300 treatment doses at about 109 plaque-forming units with endotoxin levels within human therapeutic regulatory limits. In alternative embodiments, provided are formulations or pharmaceutical preparations of bacteriophage comprising 109 PFU, 1010 PFU, 1011 PFU, or 1012 PFU or more per unit dose and endotoxin levels below about 5.5 EU·mL−1, or below about 5.0 EU·mL−1.

1 . (canceled) 
     
     
         2 . A method for purifying bacteriophage (phage) and generating a substantially endotoxin free preparation of phage, comprising:
 (a) providing a sample comprising a bacteriophage (phage), and   (b) filtering, or ultrafiltering, the sample with a dead-end microfiltration and a pressure-driven cross-flow ultrafiltration (CFF) comprising a molecular weight cut-off (MWCO) at about 100 kDa,   wherein a membrane pore size of about 100 kDa provides the ability to filter out an equivalent to a spherical particle with an about 3 nm diameter, thereby having the ability to retain substantially most phages,   wherein any material smaller than a cross-flow membrane pore passes through the membrane, while larger phage particles remain in the retentate stream, and the CFF with an about 100 kDa membrane pore size removes both free endotoxin, which are approximately 10 kDa in size, and all known exotoxins, which are typically smaller than about 30 kDa, and a membrane pore size of about 100 kDa provides an equivalent to a spherical particle with a 3 nm diameter and can retain substantially most phages,   thereby generating a substantially endotoxin free preparation of phage.   
     
     
         3 . The method of  claim 2 , wherein preparation of phage comprises endotoxin levels to below about 5.5 EU·mL-1, or below about 5.0 EU·mL-1. 
     
     
         4 . The method of  claim 2 , wherein a single production run produces at least about 300 treatment doses phage at about 10 9  PFU, 10 10  PFU, 10 11  PFU, or 10 12  PFU, or more per dose. 
     
     
         5 . The method of  claim 2 , wherein phage lysates or cultures are cooled to below about 37° C., or cooled to between about 4° C. to 37° C., at the point where phage-insensitive or phage-resistant bacteria appear in culture. 
     
     
         6 . A formulation or pharmaceutical preparation of bacteriophage (phage), comprising:
 about 10 9  PFU, 10 10  PFU, 10 11  PFU, or 10 12  PFU or more per unit dose of bacteriophage (phage) and endotoxin levels below about 5.5 EU·mL −1 , or below about 5.0 EU·mL −1 .   
     
     
         7 . The formulation or pharmaceutical preparation of  claim 6 , wherein the phage are formulated for enteral or parenteral administration, or are formulated for administration intramuscularly, orally, parenterally, by inhalation spray, nasally, topically, intrathecally, intrathecally, intracerebrally, epidurally, intracranially, vaginally or rectally. 
     
     
         8 . The formulation or pharmaceutical preparation of  claim 6 , wherein the phage are formulated as a lyophilate, a tablet, a pill, a powder, a dragee, a capsule, a liquid, a lozenge, a gel, a syrup, a slurry or a suspension. 
     
     
         9 . The formulation or pharmaceutical preparation of  claim 6 , further comprising a pharmaceutically acceptable excipient, or phage dissolved in (optionally sterile) saline, water, polyethylene glycol, propylene glycol, ethanol or oils such as safflower oil, corn oil, peanut oil, cottonseed oil or sesame oil, tragacanth gum, and/or a buffer. 
     
     
         10 . A method for treating a bacterial infection in vivo comprising administering to an individual in need thereof a formulation or pharmaceutical preparation of  claim 6 . 
     
     
         11 - 12 . (canceled) 
     
     
         13 . The method of  claim 10 , wherein the pharmaceutical preparation is administered orally, parenterally, by inhalation spray, nasally, topically, intrathecally, intrathecally, intracerebrally, epidurally, intracranially, vaginally or rectally. 
     
     
         14 . A formulation or pharmaceutical preparation of bacteriophage (phage), comprising a preparation of bacteriophage (phage) prepared by a method of  claim 2 , and having about 10 9  PFU, 10 10  PFU, 10 11  PFU, or 10 12  PFU or more per unit doses of bacteriophage (phage) and endotoxin levels below about 5.5 EU·mL −1 , or below about 5.0 EU·mL −1 . 
     
     
         15 . The formulation or pharmaceutical preparation of  claim 14 , wherein the phage are formulated for enteral or parenteral administration, or are formulated for administration intramuscularly, orally, parenterally, by inhalation spray, nasally, topically, intrathecally, intrathecally, intracerebrally, epidurally, intracranially, vaginally or rectally. 
     
     
         16 . The formulation or pharmaceutical preparation of  claim 14 , wherein the phage are formulated as a lyophilate, a tablet, a pill, a powder, a dragee, a capsule, a liquid, a lozenge, a gel, a syrup, a slurry or a suspension. 
     
     
         17 . The formulation or pharmaceutical preparation of  claim 14 , further comprising a pharmaceutically acceptable excipient, or phage dissolved in (optionally sterile) saline, water, polyethylene glycol, propylene glycol, ethanol or oils such as safflower oil, corn oil, peanut oil, cottonseed oil or sesame oil, tragacanth gum, and/or a buffer. 
     
     
         18 . A method for treating a bacterial infection in vivo comprising administering to an individual in need thereof a formulation or pharmaceutical preparation of  claim 14 . 
     
     
         19 . The method of  claim 18 , wherein the pharmaceutical preparation is administered orally, parenterally, by inhalation spray, nasally, topically, intrathecally, intrathecally, intracerebrally, epidurally, intracranially, vaginally or rectally. 
     
     
         20 . The formulation or pharmaceutical preparation, wherein the bacteriophage (phage) particles are greater than about 100 KDa in size. 
     
     
         21 . The formulation or pharmaceutical preparation of  claim 2 , wherein in step (a) the phage propagated are added during mid-log stage of bacterial growth. 
     
     
         22 . The formulation or pharmaceutical preparation of  claim 2 , wherein step (b) comprises filtering with dead-end microfiltration and cross-flow ultrafiltration (CFF).